(1) Applications of Techniques to Introduce Foreign DNA
Inserting foreign DNA into the host cell genome is a technique of significant importance that finds wide applications in many industries and medical fields.
One principal application includes the production of useful substances. In the early stage, vectors employing Escherichia coli as host cells were constructed for this purpose. These vectors are still in use today. A drawback with such system is that the produced proteins are modified in a different manner from the system of mammals or birds. This presents a problem when it is desired to produce and utilize useful proteins of humans or other animals. In particular, Escherichia coli is not ideal for producing glycoproteins since the addition of sugar chains to proteins does not take place in the E. coli system. Also, the resulting proteins often form insoluble aggregates in E. coli, which may require further treatment in order to obtain physiologically active proteins.
To avoid this problem, vectors employing yeast as hosts are used. Unlike E. coli, the addition of sugar chains occurs in yeast. However, since different types of sugar chains are added in yeast, problems may arise in terms of physiological activities. The produced proteins in yeast also tend to form aggregates as in E. coli. 
A method using viruses as an expression vector is also widely used. At present, an expression system employing baculoviruses as a vector is widely used for the purpose of obtaining physiologically active proteins in large quantities. In this system, it is more likely that physiologically active proteins are obtained as compared to the other two systems described above. In this case, however, the problem of different-type sugar chains added to glycoproteins and the problem of the formation of protein aggregates still remain. Further, the amount of a protein to be expressed may vary widely depending on the type of the protein to be produced. Thus, it is presently difficult to estimate the amount of a protein to be expressed until recombinants are constructed and the protein has actually been expressed.
In another method, plasmid vectors are introduced into animal cells to express useful proteins. This approach has an advantage that little problem arises regarding the modification of expressed proteins. Various techniques are available to introduce plasmid vectors into cells, including electroporation, calcium phosphate-DNA transfection, and the use of liposome. However, in general, the DNA introduced into cells using these techniques is not inserted into the genome and is thus lost and eventually eliminated as the cells grow. Thus, each production of a protein requires the introduction of the DNA.
When plasmid DNA is introduced into cells, recombination may occur between the plasmid DNA and the genome of the host cell, resulting in the integration of the plasmid DNA into the genome, though the frequency of the occurrence is extremely low. In general, once integrated into the genome of the host cell, the DNA will not be lost through the proliferation of the host cells. Thus, cells that can permanently express a protein of interest can be obtained by selecting the cells that the vector DNA encoding the protein has been integrated into their genome. The selection is made possible by applying an antibiotic to select the cells that are expressing a foreign gene along with a protein resistant to the antibiotic. Hence, the cells capable of expressing useful proteins are obtained selectively. However, the protein of interest is generally expressed in small amounts in the cells obtained in this manner, limiting the range of application of the method.
Production of useful proteins by the use animals has also been attempted. For this purpose, a method is attempted in which useful proteins are expressed in milk of cows, goats, hogs or the like. In another approach, useful proteins are expressed in eggs of chickens. These methods, when successfully carried out, make it possible to obtain useful proteins in substantial amounts. However, it is disadvantageous that, in the former approach, costs needed to establish transgenic (TG) animals using large mammals such as cows, goats and hogs tend to be enormous.
In the latter approach making use of chickens, it will be convenient if foreign DNA can be introduced into laid eggs as they are easy to handle. In producing mammalian TG animals, DNA is injected into nuclei of fertilized eggs using a micromanipulator before cell division is started or at a very early stage after cell division is started. This is not applicable, however, to already laid chicken eggs since cell division has already proceeded to a significant degree when the eggs are laid. Also, it is extremely complicate to introduce foreign genes into each of cells that have multiplied in number through cell division using a micromanipulator. In a recent report, fertilized eggs were collected from chickens before they underwent cell division, and plasmid DNA was microinjected into the eggs. The eggs were then artificially incubated until hatching by extrasomatic incubation. The report describes that the introduced foreign genes were successively transferred to the successive generations (Love, J., et al., Biotechnology, 12, 60-63, 1994). This approach still requires special techniques such as micromanipulation and extrasomatic incubation of fertilized chicken eggs. To overcome these problems, a method is proposed in which a foreign gene is introduced into primordial germ cells which are collected from chick embryos at an early developmental stage, and the cells are returned to early embryos in other developing chicken eggs which are then hatched. In this method, chicks whose germline cells have contained a foreign gene are obtained. When these chicks are grown and allowed to reproduce, it is expected that the foreign gene is transmitted to the descendants. However, the method has not yet been put to practical use because no effective way has been established so far to efficiently insert a foreign gene into the genome of the collected primordial germ cells.
In recent years, an approach using cloning technologies has been developed. In this approach, a gene is introduced into somatic cells collected from an animal in vitro, and the cells having genome into which the foreign gene has been integrated are selected. Using the micromanipulation technique, the nuclei collected from the selected cells are implanted into ova that have had their own nuclei removed in advance. The ova are then returned to the uterus of the female animal to obtain progeny. However, the implantation of nuclei into cells using, for example, micromanipulation technique requires special skills and, moreover, enormous costs will be needed to establish cloned animals.
Injection of DNA using a micromanipulator is also a key technique in introducing foreign DNA into primordial germ cells of chickens. However, since as many as one hundred primordial germ cells need to be returned to one early embryo in a developing chicken egg, the method using a micromanipulator is not ideal for handling the numerous primordial germ cells.
Methods for handling numerous primordial germ cells include introducing plasmid DNA by means of electroporation and using retrovirus vectors as an inherently infectious vector. In the former method, it has been reported that a gene can be introduced into primordial germ cells with a relatively high efficiency by using electroporation (Hong, Y. H., et al., Transgenic Res., 7, 247-252, 1998). However, it is still uncertain whether the gene introduced is transmitted to offspring in a stable manner since the method employs ordinary plasmid vectors. Depending on a report as to the latter case of retrovirus vectors, when the primordial germ cells into which a foreign gene has been introduced using retroviral vectors are implanted in embryos of other eggs, the foreign gene is transferred to offspring of the chickens developed from the embryos (Vick, L., et al., Proc. R. Soc. Lond. B. Biol. Sci., 251, 179-182, 1993). The method, however, is disadvantageous in that it takes a considerable amount of time to construct such vectors and in that the viruses generally do not replicate efficiently enough and viral infection is typically inefficient. Also, it is difficult to construct retrovirus vectors with a strong promoter.
While TG animals have been described in regard to the production of useful proteins, they are used in various other applications. TG mice, for example, are used to analyze functions of certain genes. TG mice into which a gene encoding a receptor for a particular pathogen has been introduced are used as a model animal for the disease caused by the pathogen. One application of TG animals includes producing animals for providing organs for organ transplantation.
Although making TG mice is an already established technique, it requires a special skill, i.e., micromanipulation, and associated equipment.
Recently, a method for producing TG animals in a more convenient manner has been proposed wherein DNA is injected directly into testis of male animals, and the males are allowed to naturally mate with female animals to introduce the foreign gene into fertilized eggs. Expression of foreign gene has been observed in early blastcysts developed from fertilized eggs which are obtained by injecting DNA into testes of male mice and allowing them to naturally mate(Ogawa S., et al., J. Reproduction and Development, 41, 379-382, 1995). However, the foreign gene introduced in this manner may not be passed on to the progeny in a stable fashion and thus the method is not effective enough to ensure the production of TG mice.
The idea of DNA vaccine has been developed recently, and significant efforts have been put into developing this concept. The principle of DNA vaccine is that immunity can be elicited in animals following the administration of DNA encoding a certain antigen under the control of promoter by injection or by particle bombardment. The basis of this idea is that DNA is more convenient to handle and is thus more advantageous in developing a new vaccine as compared to attenuated or inactivated pathogens used as vaccines, or peptides that induce protective immunity. Expression of foreign genes has actually been observed in cells of animals inoculated with plasmid DNA. Humoral immunity, as well as cellular immunity which typically is difficult to induce by inactivated vaccines, has been induced in these animals (Ulmer, J. B. et al., Science, 259, 1745-1749, 1993). However, DNA vaccines have not been put to practical use hitherto since the induction of immune responses requires significant amounts of DNA. One reason for this is that the DNA taken up by cells might be eliminated in a short period of time. To cope with this, it is investigated to utilize retroviruses, which become a provirus, as a vector. In particular, it is proposed to use as a vaccine a vector into which env/rev genes of HIV, a virus responsible for AIDS, have been incorporated (Irwin, M. J., et al., J. Virol., 68, 5036-5044, 1994). The use of retroviruses as a vector is accompanied by several problems, however. (This will be described in detail later.)
Techniques for integration of a foreign gene into the genome of host cells are used as a gene therapy to treat incurable diseases of humans. A primary application of the gene therapy concerns treating genetic diseases. Genetic diseases are defined as the diseases caused by defects or mutation in some important genes. Thus, by introducing normal genes that compensate for the defective or mutated genes, the symptoms may be ameliorated or eliminated. Although the number of genetic diseases to which the gene therapy can be applied is limited as it is literally impossible to introduce a gene into all of the cells, the gene therapy has proven to be successful in treating some genetic diseases including adenosine deaminase (ADA) deficiency.
A second application of the gene therapy concerns treating cancers as described below. A tumor suppressor gene or a gene that expresses antisense RNA of an oncogene is introduced to suppress the development of cancers. This approach is thought to be one of the most promising applications of the therapy. In another approach, a gene encoding a cytokine is introduced into immunocompetent cells or cancer cells, or a gene associated with a major histocompatibility complex (MHC) is introduced into cancer cells in order to enhance immune activities of host and consequently suppress cancers. Also, a gene that makes cells resistant to anticancer drugs may be introduced to protect the cells, such as bone marrow stem cells, that are otherwise susceptible to the anticancer drugs. This intends to enable administration of anticancer drugs in larger dosages.
A third application of the gene therapy concerns treating infectious diseases. The diseases addressed by this approach include AIDS and hepatitis C where pathogens relatively grow slowly and evade the immune system of the hosts. A gene capable of suppressing the expression of the genes that are essential to the growth of a pathogen (e.g., a gene encoding antisense RNA or a gene encoding a ribozyme that specifically cleaves viral RNA) may also be introduced into the target cells of the pathogen, such that the cells that have contained the gene will be protected against the pathogens. As such, the concept of intracellular immunization, meaning anti-disease activity on a cell basis, has recently been proposed and much effort has been made to develop the idea (Baltimore, D., Nature, 335, 395-396, 1988).
(2) Methods for Inserting DNA Using Retrovirus Vectors or the Like.
A method for inserting foreign DNA into a genome involves introducing a plasmid vector having a foreign DNA and a drug resistant gene into cells, and then, by using the drug, selecting the cells into the genome of which the foreign DNA has been incorporated. While this method is effective to a certain degree, its use is limited to certain applications such as cultured cells due to extremely low efficiencies.
Another method for inserting foreign DNA into the genome, which is often used to produce TG animals, involves directly injecting an amount of DNA into nuclei. This method achieves a higher probability that the foreign DNA is integrated into the genome since a large quantity of foreign DNA is directly injected into nuclei. The method, however, is not ideal for handling a number of cells at a time because it requires special equipment such as a micromanipulator as well as skills to use such equipment and because cells have to be handled individually, making the method extremely difficult.
Accordingly, the most popular method for inserting any DNA sequence of interest into the genome of host cells, at present, seems to be the one employing retroviruses. Retroviruses are single-stranded RNA viruses, and their genomic RNA has the structure of which type is essentially a messenger RNA (mRNA) that has a cap structure at 5′ end and a poly-A tail at 3′ end. Upon infection, viral particles bind to receptors of the host cell via a specific binding. The virus envelope and the cell membrane fuses to release a pre-integration complex into the cytoplasm. The complex includes reverse transcriptase and integrase, each originating from the viral particle, in addition to viral RNA. The reverse transcriptase catalyzes the synthesis of a double-stranded, linear DNA using the viral RNA as a template. Through this process, a long repetitive sequence, termed the long terminal repeat(LTR), which is not found in the viral RNA, is formed at each end of the DNA strand. This double-stranded linear DNA, together with integrase, forms a complex and moves into a nucleus where it subsequently circularizes. The resulting circular DNA is integrated into the genome of the host cell with the help of integrase (Molecular Biology of Genes, 4th ed. Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz, J. A., Weiner, A. M., Japanese translation prepared by Kenichi Matsubara, Keiko Nakamura, Kinichiro Miura, published by Toppan Ltd., 1988). However, there is a report suggesting that the precursor of the integration into the host genome is not the circular DNA but linear double stranded DNA (Brown, P. O., et al., Proc. Natl. Acad. Sci. USA., 86, 2525-2529, 1989). It is thought that the integrase that plays a role here is not newly produced from the viral genome via transcription and translation of the genome following the infection. Rather, the enzyme seems to be of virion-origin and is thought to be brought into the host cell upon infection. The localization of integrase into a nucleus and functioning of the enzyme appear to be occurring while integrase is bound to the viral genome.
A report suggests that the integration into the genome of host cells occurs at a site where LTRs at each end of the linear double-stranded DNA are joined to one another as the linear double-stranded DNA circularizes (Panganiban, A. T, and Temin, H. M., Cell, 36, 673-679, 1984). Panganiban, A. T, and Temin, H. M. (Nature, 306, 155-160. 1983) and Duyk, G. et al. (J. Virol., 56, 589-599, 1985) describe the recognition sequences of integrase.
The form of virus that is integrated into the genome of a host cell is referred to, as a provirus. RNA polymerase II of the host cell recognizes a promoter in the LTR of a provirus, initiating the transcription of the retroviral genome into RNA. A part of the transcribed RNA serves as viral RNA while the other part of the RNA undergoes splicing and serves as mRNA to synthesize viral proteins. As described above, retroviruses are integrated into the genome of host cells and become a provirus in their life cycle. This characteristic makes retroviruses a vector for inserting a foreign gene into the genome of host cells.
Retroviruses carry the genes gag, pol, and env. When these genes are defective, the retroviruses can still infect the host cell but are no longer capable of producing virions of the next generation. This implies the possibility that a retrovirus, in which one or more of these genes have been made defective and a foreign gene has been inserted instead, may be used as a safe vector that can insert a foreign gene into the genome of the host cell but will no longer produce infectious virions.
However, a problem exists concerning the safety of the retroviral vectors. In order to enable these vectors to replicate, cells expressing the viral proteins for the defective genes (i.e., helper cells) are established and used. Thus, there is a chance, though small, that a homologous recombination occurs between the viral genes present in the helper cell and the vector plasmid carrying a foreign gene, resulting in replicant competent retroviruses (RCRs) that have regained the ability to replicate. In fact, there is a report in which these viruses have caused the T-cell lymphoma in monkeys (Donahue, R. E., et al., J. Exp. Med., 176, 1125-1135, 1992).
While retroviral vectors are, in principle, ideal for inserting a foreign gene into the genome of host cells, some drawbacks exist with this approach such as follows:
(i) It is difficult to obtain high-titer viruses. The highest titer of the viruses that can be obtained is typically around 105 to 107 colony forming unit (cfu)/ml.
Also, polypeptides such as SU polypeptides constituting envelope proteins that is involved in the specific binding to the receptors of the host cell will be lost during the process of concentration of virions, for example, by centrifugation. Thus, it is difficult to obtain infectious viral particles in high concentrations. This is one of the reasons for the difficulty in obtaining high-titer viruses.
(ii) The efficiency of viral infection is low. This, in addition to the difficulty in obtaining high-titer viruses, also makes it difficult to achieve the gene introduction with sufficient efficiency.
(iii) It is difficult to construct recombinants incorporating a strong promoter. This is a problem when high levels of foreign gene expression are desired.
(iv) Generally, it takes considerable amount of time to establish practical viral vectors.
(v) In many cases, the infection efficiency of retroviral vectors and the expression efficiency of the introduced gene are low.
(vi) Viruses tend to be inactivated by complements in vivo.
Advantages and disadvantages of the vectors for introducing a gene that are currently in use are summarized in Table 1 below.
TABLE 1Comparison between vectors for gene introductionAdvantagesDisadvantagesRETROVIRAL* They are integrated* Insertion of the vector mayVECTORSinto the genome ofcause mutation in the gene of thehost cells.host cells(2).* They can be used to* When viruses belonging tointroduce a gene intothe oncovirus subfamilyvarious types of cellsare used as a vector,including blood cells.they cannot use for gene* They can be readilytransfer into non-dividing cells.constructed by using* Low expression efficiency ofhelper cells(1).introduced gene (Constructingviral vectors with a strongpromoter is difficult).* It is difficult to obtainhigh-titer viruses.* Infectious viruses may arise asa result of homologousrecombination(3).ADENO-* They can transfer a* They are not inserted into hostVIRALforeign gene with highgenome, and expression ofVECTORSefficiency.introduced foreign gene is* They may be usedtransient.for gene transfer* They exhibit strongin vivo.antigenicity and thus are not* They may be usedsuitable for repeatedfor gene transferadministration to livinginto non-dividingorganisms.cells.* Constructing vectors involves* High-titer vectorscomplicated process.can be prepared.* They exhibit cytotoxicity.ADENO-* They can transfer a* Site-specific integration,ASSOCIATEDforeign gene intowhich is observed in wild typeVIRALdividing andviruses, does not occur.VECTORSnon-dividing cells* Genes equal to or larger thanwith relatively4.5 kb in size can not be insertedhigh efficiency.into vectors.* They are integrated* Construction of vectorsinto host genome,involves complicated process.though efficiency is* Mass-production of the vectorsnot always high.is difficult.* They do not exhibit* Low titerpathogenicityor cytotoxicity.(1)Cells carrying plasmids that encode viral proteins but do not have a packaging signal. (2)With regard to the possibility of the transformation into cancer, which is of particular concern, the chance of occurrence seems to be very small due to the involvement of several genes in the transformation. No case of cancer development caused by retroviral vectors has ever been reported. (3)Occurrence of T-lymphoma was reported in monkeys in 1991. 
Among reports concerning integrase of retroviruses is the one by Tanaka et al (Shoji-Tanaka, A., et al., Biochem. Biophys. Res. Commun., 203, 1756-1764, 1994). In this report, it is observed that the efficiency of DNA introduction into cells was increased by several times by introducing into cells purified integrase of bovine leukemia viruses. The integrase was expressed in E. coli and purified. Using liposome carriers, the purified integrase was introduced along with plasmids incorporating an integrase recognition site. The degree of this increase in the efficiency of DNA introduction into cells was varied depending on whether or not a linking site of LTRs and sequences adjacent to the linking site were contained in the plasmid. Determination of cleavage sites in the plasmid DNA integrated into the genome using the southern hybridization technique indicated that integration occured at the site where the LTR linking site exists when the plasmids were introduced into cells with integrase, whereas recombination seems to have occurred in the plasmid in the regions other than that which includes the LTR linking site when plasmids alone were introduced into cells without integrase.
In this report, the integrase was first mixed with the DNA, and the mixture was then incubated at a room temperature for thirty minutes. This is supposed to facilitate the formation of complexes of integrase and the DNA by utilizing integrase's ability to bind to DNA. Thus, following the introduction into cells by means of liposome carriers, the integrase and the plasmid are not transferred into a nucleus individually, but they are transferred into a nucleus as a complex. Accordingly, the insertion of DNA into the genome is carried out in this system by making use of the life cycle of retroviruses described above.
However, Mochii reports that no increase was observed in the efficiency with which a foreign gene was integrated into the genome of cells when the above-described method by Tanaka et al was applied to fertilized eggs of chickens (Protein, Nucleic acid and Enzyme, 40, 2265-2273, 1995).